Glucagon analogues

ABSTRACT

The invention provides materials and methods for promoting weight loss or preventing weight gain, and in the treatment of diabetes, metabolic syndrome and associated disorders. In particular, the invention provides novel glucagon analogue peptides effective in such methods. The peptides may mediate their effect by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage filing under 35 U.S.C. §371of international application PCT/GB2008/004121, filed Dec. 15, 2008.

FIELD OF THE INVENTION

The present invention relates to glucagon analogues and their medicaluse, for example in the treatment of excess food intake, obesity andexcess weight.

BACKGROUND OF THE INVENTION

Preproglucagon is a 158 amino acid precursor polypeptide that isdifferentially processed in the tissues to form a number of structurallyrelated proglucagon-derived peptides, including glucagon (Glu),glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), andoxyntomodulin (OXM). These molecules are involved in a wide variety ofphysiological functions, including glucose homeostasis, insulinsecretion, gastric emptying and intestinal growth, as well as regulationof food intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon and has the sequenceHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1). Oxyntomodulin (OXM) is a 37 amino acid peptide whichincludes the complete 29 amino acid sequence of glucagon with anoctapeptide carboxyterminal extension (amino acids 82 to 89 ofpre-proglucagon, having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala(SEQ ID NO: 2) and termed “intervening peptide 1” or IP-1; the fullsequence of human oxyntomodulin is thusHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala)(SEQ ID NO: 3). The major biologically active fragment of GLP-1 isproduced as a 30-amino acid, C-terminally amidated peptide thatcorresponds to amino acids 98 to 127 of pre-proglucagon.

Glucagon helps maintain the level of glucose in the blood by binding toglucagon receptors on hepatocytes, causing the liver to releaseglucose—stored in the form of glycogen—through glycogenolysis. As thesestores become depleted, glucagon stimulates the liver to synthesizeadditional glucose by gluconeogenesis. This glucose is released into thebloodstream, preventing the development of hypoglycemia.

OXM is released into the blood in response to food ingestion and inproportion to meal calorie content. OXM has been shown to suppressappetite and inhibit food intake in humans (Cohen et al, Journal ofEndocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). Inaddition to those anorectic effects, which are similar to those ofGLP-1, OXM must also affect body weight by another mechanism, since ratstreated with oxyntomodulin show less body weight gain than pair-fed rats(Bloom, Endocrinology 2004, 145, 2687). Treatment of obese rodents withOXM also improves their glucose tolerance (Parlevliet et al, Am JPhysiol Endocrinol Metab, 294, E142-7, 2008) and suppresses body weightgain (WO 2003/022304).

OXM activates both the glucagon receptor and the GLP-1 receptor with atwo-fold higher potency for the glucagon receptor over the GLP-1receptor, but is less potent than native glucagon and GLP-1 on theirrespective receptors. Glucagon is also capable of activating bothreceptors, though with a strong preference for the glucagon receptorover the GLP-1 receptor. GLP-1 on the other hand is not capable ofactivating the glucagon receptor. The mechanism of action ofoxyntomodulin is not well understood. In particular, it is not knownwhether the effects of the hormone are mediated exclusively through theglucagon receptor and the GLP-1 receptor, or through one or more as-yetunidentified receptors.

Other peptides have been shown to bind and activate both the glucagonand the GLP-1 receptor (Hjort et al, Journal of Biological Chemistry,269, 30121-30124, 1994) and to suppress body weight gain and reduce foodintake (WO 2006/134340; WO 2007/100535; WO 2008/101017).

Obesity, classified is a globally increasing health problem and isassociated with various diseases, particularly cardiovascular disease(CVD), type 2 diabetes, obstructive sleep apnea, certain types ofcancer, and osteoarthritis. As a result, obesity has been found toreduce life expectancy. According to 2005 projections by the WorldHealth Organization there are 400 million adults (age>15) classified asobese worldwide. In the US, obesity is now believed to be thesecond-leading cause of preventable death after smoking.

The rise in obesity drives an increase in diabetes, and approximately90% of people with type 2 diabetes may be classified obese. There are246 million people worldwide with diabetes, and by 2025 it is estimatedthat 380 million will have diabetes. Many have additional cardiovascularrisk factors including high/aberrant LDL and triglycerides and low HDL.

People with diabetes are 2 to 4 times more likely to developcardiovascular disease than people without diabetes, making it the mostcommon complication of diabetes. Cardiovascular disease accounts forabout 50% of the mortality in people with diabetes. Young adults withdiabetes have rates of coronary heart disease (CHD) 12-40 times higherthan those in young adults without diabetes and together with the highincidence and prevalence of obesity and type 2 diabetes, the morbidityand mortality rates relating to these metabolic disorders underscore themedical need for efficacious treatment options

Accordingly, there is a strong medical need for treating obesity andimproving glucose tolerance.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound having the formulaR¹-X-Z-R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide which has the formula I:

(SEQ ID NO: 4) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-Arg-Alaor differs from formula I at up to 4 of the following positions whereby,if different from formula I:the residue at position 2 is selected from: Aib, D-Ser;the residue at position 12 is selected from: Leu, Arg, Dpu, Dpr, Orn;the residue at position 16 is selected from: Arg, His, Lys, Glu, Asp;the residue at position 17 is selected from: Arg, Leu, Dpu, Dpr, Orn;the residue at position 18 is selected from: Arg, Lys, His, Ser, Tyr;the residue at position 20 is selected from: Gln, Lys, Arg, Glu, Asp;the residue at position 21 is Glu;the residue at position 24 is selected from: Gln, Leu, Ala, Lys, Arg,Asp;the residue at position 27 is selected from: Met, Cys, Lys, Arg, Glu;the residue at position 28 is selected from: Asn, Ser, Lys, Glu, Ala,Leu, Asp; andthe residue at position 29 is selected from: Thr, Glu, Lys;and Z is absent or a peptide sequence of 1-20 amino acid units selectedfrom the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys,Arg, Dbu, Dpr and Orn;or a pharmaceutically acceptable salt thereof.

In certain embodiments of the first aspect, X may differ from formula Iat up to 4 of the following positions whereby, if different from formulaI:

the residue at position 2 is selected from: Alb, D-Ser;

the residue at position 16 is selected from: Arg, His, Lys, Glu;

the residue at position 17 is selected from: Arg, Leu;

the residue at position 18 is selected from: Arg, Lys, His, Ser, Tyr;

the residue at position 20 is selected from: Gln, Lys, Arg, Glu;

the residue at position 24 is selected from: Gln, Leu, Ala, Lys, Arg;

the residue at position 27 is selected from: Met, Cys, Lys, Arg, Glu;

the residue at position 28 is selected from: Asn, Ser, Lys, Glu, Ala,Leu; and

the residue at position 29 is selected from: Thr, Glu, Lys.

In certain embodiments of the first aspect, X may differ from formula Iat up to 4 of the following positions whereby, if different from formulaI:

the residue at position 2 is selected from: Aib, D-Ser;

the residue at position 16 is selected from: Arg, His, Lys, Glu, Gly;

the residue at position 24 is selected from: Gln, Leu, Ala, Lys, Arg;

the residue at position 27 is selected from: Met, Cys, Lys, Arg, Glu;

the residue at position 28 is selected from: Asn, Ser, Lys, Glu, Ala,Leu; and

the residue at position 29 is selected from: Thr, Glu, Lys.

While maintaining consistency with the definitions of the first aspectabove, it may be desirable that X comprises one or more of the followingsets of residues:

17-Lys, 18-Ala; 17-Leu, 18-Ala;

17-Lys, 18-Ala, 20-His;

17-Leu, 18-Ala, 20-His;

17-Lys, 18-Ala, 24-Glu;

17-Leu, 18-Ala, 24-Glu;

17-Lys, 18-Ala, 27-Leu;

17-Leu, 18-Ala, 27-Leu;

17-Lys, 18-Ala, 29-Ala;

17-Leu, 18-Ala, 29-Ala;

17-Lys, 18-Ala, 27-Leu, 29-Ala;

17-Leu, 18-Ala, 27-Leu, 29-Ala;

17-Lys, 18-Ala, 27-Leu, 28-Arg, 29-Ala;

17-Leu, 18-Ala, 27-Leu, 28-Arg, 29-Ala;

24-Glu, 28-Arg;

24-Glu, 28-Arg, 27-Leu;

24-Glu, 28-Arg, 27-Leu, 29-Ala;

27-Leu, 28-Arg, 29-Ala;

29-Ala;

20-Arg, 24-Arg, 27-Lys, 28-Leu;

17-Arg;

18-Arg;

20-Gln;

24-Gln;

27-Met, 28-Asn, 29-Thr; or

24-Lys

and combinations thereof.

For example, X may have the sequence:

(SEQ ID NO: 5) HSQGTFTSDYSKYLDSKAARDFVRWLKLA; (SEQ ID NO: 12)HSQGTFTSDYSKYLDSRAAHDFVEWLLRA; (SEQ ID NO: 13)HSQGTFTSDYSKYLDSKRAHDFVEWLLRA; (SEQ ID NO: 8)HSQGTFTSDYSKYLDSKAAQDFVEWLLRA; (SEQ ID NO: 9)HSQGTFTSDYSKYLDSKAAHDFVQWLLRA; (SEQ ID NO: 10)HSQGTFTSDYSKYLDSKAAHDFVEWLMNT; (SEQ ID NO: 11)HSQGTFTSDYSKYLDSKAAHDFVKWLLRA; (SEQ ID NO: 6)H-DSer-QGTFTSDYSKYLDSKAAHDFVEWLLRA; (SEQ ID NO: 7)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA; (SEQ ID NO: 18)HSQGTFTSDYSKYLDSKAAKDFVEWLLRA; (SEQ ID NO: 19)HSQGTFTSDYSKYLDKKAAHDFVEWLLRA or (SEQ ID NO: 20)HSQGTFTSDYSKYLDSKAAQDFVEWLLRA.

In a second aspect, the invention provides a compound having the formulaR¹-X-Z-R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

X is a peptide which has the formula

(SEQ ID NO: 4) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-Arg-Alaor differs from formula I at up to 5 of the following positions whereby,if different from formula I:the residue at position 2 is selected from: Aib, D-Ser;the residue at position 16 is selected from: Arg, His, Lys, Glu;the residue at position 17 is: Arg, Leu, Dpu, Dpr, Orn;the residue at position 20 is selected from: Gln, Lys, Arg, Glu, Asp;the residue at position 21 is Glu;the residue at position 24 is selected from: Gln, Leu, Ala, Lys, Arg,Asp;the residue at position 27 is selected from: Met, Cys, Lys, Arg, Glu;the residue at position 28 is selected from: Asn, Ser, Lys, Glu, Ala,Leu, Asp; andthe residue at position 29 is selected from: Thr, Glu, Lys;and Z is absent or a peptide sequence of 1-20 amino acid units selectedfrom the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys,Arg, Dbu, Dpr and Orn;or a pharmaceutically acceptable salt thereof.

In certain embodiments of the second aspect, X may differ from formula Iat up to 4 of the following positions whereby, if different from formulaI:

the residue at position 2 is selected from: Aib, D-Ser;

the residue at position 16 is selected from: Arg, His, Lys, Glu, Gly;

the residue at position 17 is selected from: Arg, Leu;

the residue at position 18 is selected from: Arg, Lys, His, Ser, Tyr;

the residue at position 20 is selected from: Gln, Lys, Arg, Glu;

the residue at position 24 is selected from: Gln, Leu, Ala, Lys, Arg;

the residue at position 27 is selected from: Met, Cys, Lys, Arg, Glu;

the residue at position 28 is selected from: Asn, Ser, Lys, Glu, Ala,Leu; and

the residue at position 29 is selected from: Thr, Glu, Lys.

While maintaining consistency with the definitions of the second aspectabove, it may be desirable that X comprises any of the sets of residuesdescribed above in relation to the first aspect, or one or more of thefollowing sets of residues:

20-Gln, 24-Gln, 27-Met, 28-Asn, 29-Thr; or

17-Leu, 20-Gln, 24-Gln, 28-Asn, 29-Thr.

HSQGTFTSDYSKYLDSKAAQDFVQWLMNT (SEQ ID NO: 15) orHSQGTFTSDYSKYLDSLAAQDFVQWLLNT. (SEQ ID NO: 14)

The invention further provides a nucleic acid (which may be DNA or RNA)encoding a compound of the invention, an expression vector comprisingsuch a nucleic acid, and a host cell containing such a nucleic acid orexpression vector.

In a further aspect, the present invention provides a compositioncomprising a glucagon analogue peptide as defined herein, or a salt orderivative thereof, a nucleic acid encoding such a glucagon analoguepeptide, an expression vector comprising such a nucleic acid, or a hostcell containing such a nucleic acid or expression vector, in admixturewith a carrier. In preferred embodiments, the composition is apharmaceutically acceptable composition and the carrier is apharmaceutically acceptable carrier. The glucagon peptide analogue maybe a pharmaceutically acceptable acid addition salt of the glucagonanalogue.

The compounds described find use in preventing weight gain or promotingweight loss. By “preventing” is meant inhibiting or reducing weight gainwhen compared to the absence of treatment, and is not necessarily meantto imply complete cessation of weight gain. The peptides may cause adecrease in food intake and/or increased energy expenditure, resultingin the observed effect on body weight. Independently of their effect onbody weight, the compounds of the invention may have a beneficial effecton glucose tolerance and circulating cholesterol levels, being capableof lowering circulating LDL levels and increasing HDL/LDL ratio. Thusthe compounds of the invention can be used for direct or indirecttherapy of any condition caused or characterised by excess body weight,such as the treatment and/or prevention of obesity, morbid obesity,obesity linked inflammation, obesity linked gallbladder disease, obesityinduced sleep apnea. They may also be used for the treatment ofmetabolic syndrome, insulin resistance, glucose intolerance, type-2diabetes, hypertension, atherogenic dyslipidimia, atherosclerois,arteriosclerosis, coronary heart disease, or stroke. Their effects inthese conditions may be as a result of or associated with their effecton body weight, or may be independent thereof.

Thus the invention provides use of a compound of the invention in thetreatment of a condition as described above, in an individual in needthereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for the treatment of a condition asdescribed above.

As already described, the invention extends to expression vectorscomprising the above-described nucleic acid sequence, optionally incombination with sequences to direct its expression, and host cellscontaining the expression vectors. Preferably the host cells are capableof expressing and secreting the compound of the invention. In a stillfurther aspect, the present invention provides a method of producing thecompound, the method comprising culturing the host cells underconditions suitable for expressing the compound and purifying thecompound thus produced.

The invention further provides a nucleic acid of the invention, anexpression vector of the invention, or a host cell capable of expressingand secreting a compound of the invention, for use in a method ofmedical treatment. It will be understood that the nucleic acid,expression vector and host cells may be used for treatment of any of thedisorders described herein which may be treated with the compoundsthemselves. References to a therapeutic composition comprising acompound of the invention, administration of a compound of theinvention, or any therapeutic use thereof, should therefore be construedto encompass the equivalent use of a nucleic acid, expression vector orhost cell of the invention except where the context demands otherwise.

DESCRIPTION OF THE FIGURES

FIG. 1. Effect of ZP2669 on oral glucose tolerance in db/db mice. Db/dbmice were fasted overnight and an initial blood sample (fasting bloodglucose level) taken just before administration (i.p.) of vehicles orZP2669 (45 nmol/kg). Fifteen minutes later an oral dose of glucose (1g/kg in 5 ml/kg) was given and BG levels were measured at t=30 min, t=60min, t=120 min and t=240 min. Difference from baseline (t=0) wascalculated for each time point and AUC₀₋₂₄₀ min values were determined.ZP2669 significantly improved glucose tolerance in diabetic db/db mice.

FIG. 2. Effect of ZP2669 on food intake in mice. Groups of stratifiedmice stratified after body weight were fasted overnight and treated withPYY₃₋₃₆ (30 nmol/kg) (internal positive control), glucagon (500nmol/kg), ZP2669 (500 nmol/kg) or vehicle. After one hour pre-weighedfood was introduced to the mice and food intake measured by weighing theremaining food after one hour and expressed relative to body weight (mgfood/g BW). PYY(3-36) showed an anorectic effect as expected fromprevious findings. ZP2669 (500 nmol/kg) significantly decreased foodintake during the first hour following injection of peptide. Glucagonhad no effect on food intake.

FIG. 3. Effect of 28 day treatment with ZP2669 on body weight in dietinduced obese (DIO) mice. C57BI/6 male mice were put on high fat diet(HFD) and treated (b.i.d.; s.c.) with ZP2669 (500 nmol/kg) or vehicle. Anon-obese control group maintained on regular chow was treated withvehicle (CHOW) in the same treatment regimen as the DIO groups. Bodyweights were recorded daily and used to administer the bodyweight-corrected doses of peptide throughout the study. ZP2669 decreasedbody weight gain to a level similar to that observed with chow feeding.

FIG. 4. Effect of dual GIuGLP-1 agonist treatment of DIO mice for 4weeks (b.i.d.) on concentrations of LDL cholesterol. The effect of OXM(p=0.002) and ZP2669 (p=0.0006) were significant compared to vehicle.

FIG. 5. Effect of dual GIuGLP-1 agonist treatment of DIO mice for 4weeks (b.i.d.) on HDL/LDL ratios. PBS (pH 7.4) was the vehicle used forOXM, exendin-4 while acetate (pH 5.0) was the vehicle used for ZP2669.The effect of OXM (P=0.2%) and ZP2669 (P=0.05%) were significantcompared to vehicle.

FIG. 6: The chemical modification of 17-Lys in compound ZP2872.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the conventional one letter and threeletter codes for naturally occurring amino acids are used, as well asgenerally accepted three letter codes for other amino acids, such as Aib(α-aminoisobutyric acid), Orn (ornithine), Dbu (2,4 diaminobutyric acid)and Dpr (2,3-diaminopropanoic acid).

The term “native glucagon” refers to native human glucagon having thesequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH(SEQ ID NO: 1).

The terms “oxyntomodulin” and “OXM” refer to native human oxyntomodulinhaving the sequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-OH(SEQ ID NO: 3).

The invention provides compounds as defined above. For the avoidance ofdoubt, in the definitions provided herein, it is generally intended thatthe sequence of X only differs from Formula I at those positions whichare stated to allow variation. Amino acids within the sequence X can beconsidered to be numbered consecutively from 1 to 29 in the conventionalN-terminal to C-terminal direction. Reference to a “position” within Xshould be construed accordingly, as should reference to positions withinnative human glucagon and other molecules.

The compounds of the invention may carry one or more intramolecularbridge within the peptide sequence X. Each such bridge is formed betweenthe side chains of two amino acid residues of X which are typicallyseparated by three amino acids in the linear sequence of X (i.e. betweenamino acid A and amino acid A+4).

More particularly, the bridge may be formed between the side chains ofresidue pairs 12 and 16, 16 and 20, 17 and 21, 20 and 24, or 24 and 28.The two side chains can be linked to one another through ionicinteractions, or by covalent bonds. Thus these pairs of residues maycomprise oppositely charged side chains in order to form a salt bridgeby ionic interactions. For example, one of the residues may be Glu orAsp, while the other may be Lys or Arg. The pairings of Lys and Glu andLys and Asp, may also be capable of reacting to form a lactam ring.Likewise, a Tyr and a Glu or a Tyr and a Asp are capable of forming alactone ring.

In particular, the residues at positions 20 and 24 may be capable offorming an intramolecular bridge. Examples of suitable pairs of residuesat these positions include:

20-Asp, 24-Lys;

20-Glu, 24-Lys;

20-Asp, 24-Arg;

20-Glu, 24-Arg;

20-Lys, 24-Asp;

20-Arg, 24-Asp;

20-Lys, 24-Glu; and

20-Arg, 24-Glu.

Without wishing to be bound by any particular theory, it is believedthat such intramolecular bridges stabilise the alpha helical structureof the molecule and so increase potency and/or selectivity at the GLP-1receptor and possibly also the glucagon receptor.

A substitution at position 12 (e.g. of Arg or Leu) may increase potencyand/or selectivity at the GLP-1 receptor.

The presence of a basic amino acid (Arg, Lys or His) at position 16 mayenhance the molecule's solubility and may enhance bioavailability.

The arginine residues at positions 17 and 18 of native glucagon appearto provide significant selectivity for the glucagon receptor. A numberof substitutions at one or more of these positions can increase potencyand/or selectivity for both the GLP-1 receptor and the glucagonreceptor. Lys, or Leu at position 17 may increase potency at theglucagon receptor. Lys may be particularly effective, especially whenthere is a negatively charged residue (e.g. Asp) at position 21, becauseof the potential to form an intramolecular bridge. A hydrophobic orsmall hydrophilic residue (e.g. Ala or Ser) at position 18 may increasepotency at both GLP-1 and glucagon receptors.

The residues at positions 27, 28 and 29 of native glucagon appear toprovide significant selectivity for the glucagon receptor. Substitutionsat one, two, or all three of these positions with respect to the nativeglucagon sequence may increase potency at and/or selectivity for theGLP-1 receptor, potentially without significant reduction of potency atthe glucagon receptor. Particular examples include Leu at position 27,Arg at position 28 and Ala at position 29. 28-Arg may be particularlypreferred when there is a negatively charged amino acid (e.g. Glu) atposition 24 with which it can form an intramolecular bridge, which mayincrease its effect on potency at the GLP-1 receptor.

Substitution of the naturally-occurring Met residue at position 27 (e.g.with Leu, Lys or Glu) also reduces the potential for oxidation, soincreasing the chemical stability of the compounds.

Substitution of the naturally-occurring Asn residue at position 28 (e.g.by Arg or Leu) also reduces the potential for deamidation in acidicsolution, so increasing the chemical stability of the compounds.

Potency and/or selectivity at the GLP-1 receptor may also be increasedby introducing residues that are likely to form an amphipathic helicalstructure, potentially without significant loss of potency at theglucagon receptor. This may be achieved by introduction of chargedresidues at one or more of positions 16, 20, 24, and 28. Thus theresidues of positions 24 and 28 may all be charged, the residues atpositions 20, 24, and 28 may all be charged, or the residues atpositions 16, 20, 24, and 28 may all be charged. For example, theresidue at position 20 may be His or Arg, particularly His. The residueat position 24 may be Glu, Asp Lys or Arg, particularly Glu. The residueat position 28 may be Arg.

Substitution of one or both of the naturally-occurring Gln residues atpositions 20 and 24 also reduces the potential for deamidation in acidicsolution, so increasing the chemical stability of the compounds.

The compound may comprise a C-terminal peptide sequence Z of 1-20 aminoacids, for example to stabilise the conformation and/or secondarystructure of the glucagon analogue peptide, and/or to make the glucagonanalogue peptide more resistant to enzymatic hydrolysis, e.g. asdescribed in WO99/46283.

When present, Z represents a peptide sequence of 1-20 amino acidresidues, e.g. in the range of 1-15, more preferably in the range of1-10 in particular in the range of 1-7 amino acid residues, e.g., 1, 2,3, 4, 5, 6 or 7 amino acid residues, such as 6 amino acid residues. Eachof the amino acid residues in the peptide sequence Z may independentlybe selected from Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Mg, Dbu (2,4diaminobutyric acid), Dpr (2,3-diaminopropanoic acid) and Orn(ornithine). Preferably, the amino acid residues are selected from Ser,Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn, more preferably may beselected exclusively from Glu, Lys, and Cys. The above-mentioned aminoacids may have either D- or L-configuration, but preferably have anL-configuration. Particularly preferred sequences Z are sequences offour, five, six or seven consecutive lysine residues (i.e. Lys₃, Lys₄(SEQ ID NO:21), Lys₅ (SEQ ID NO:22), Lys₆ (SEQ ID NO:23) or Lys₇ (SEQ IDNO:24)), and particularly five or six consecutive lysine residues. Otherexemplary sequences of Z are shown in WO 01/04156. Alternatively theC-terminal residue of the sequence Z may be a Cys residue. This mayassist in modification (e.g. PEGylation) of the compound. In suchembodiments, the sequence Z may, for example, be only one amino acid inlength (i.e. Z=Cys) or may be two, three, four, five, six or even moreamino acids in length. The other amino acids therefore serve as a spacerbetween the peptide X and the terminal Cys residue.

The peptide sequence Z has no more than 25% sequence identity with thecorresponding sequence of the IP-1 portion of human OXM (which has thesequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).

“Percent (%) amino acid sequence identity” of a given peptide orpolypeptide sequence with respect to another polypeptide sequence (e.g.IP-1) is calculated as the percentage of amino acid residues in thegiven peptide sequence that are identical with corresponding amino acidresidues in the corresponding sequence of that other polypeptide whenthe two are aligned with one another, introducing gaps for optimalalignment if necessary. % identity values may be determined byWU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)).WU-BLAST-2 uses several search parameters, most of which are set to thedefault values. The adjustable parameters are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11. A% amino acid sequence identity value is determined by the number ofmatching identical residues as determined by WU-BLAST-2, divided by thetotal number of residues of the reference sequence (gaps introduced byWU-BLAST-2 into the reference sequence to maximize the alignment scorebeing ignored), multiplied by 100.

Thus, when Z is aligned optimally with the 8 amino acids of IP-1, it hasno more than two amino acids which are identical with the correspondingamino acids of IP-1.

One or more of the amino acid side chains in the compound of theinvention may be conjugated to a lipophilic substituent. The lipophilicsubstituent may be covalently bonded to an atom in the amino acid sidechain, or alternatively may be conjugated to the amino acid side chainby a spacer. The amino acid may be part of the peptide X, or part of thepeptide Z.

Without wishing to be bound by theory, it is thought that the lipophilicsubstituent binds albumin in the blood stream, thus shielding thecompounds of the invention from enzymatic degradation which can enhancethe half-life of the compounds. The spacer, when present, is used toprovide a spacing between the compound and the lipophilic substituent.

The lipophilic substituent may be attached to the amino acid side chainor to the spacer via an ester, a sulphonyl ester, a thioester, an amideor a sulphonamide. Accordingly it will be understood that preferably thelipophilic substituent includes an acyl group, a sulphonyl group, an Natom, an O atom or an S atom which forms part of the ester, sulphonylester, thioester, amide or sulphonamide. Preferably, an acyl group inthe lipophilic substituent forms part of an amide or ester with theamino acid side chain or the spacer.

The lipophilic substituent may include a hydrocarbon chain having 4 to30 C atoms. Preferably it has at least 8 or 12 C atoms, and preferablyit has 24 C atoms or fewer, or 20 C atoms or fewer. The hydrocarbonchain may be linear or branched and may be saturated or unsaturated. Itwill be understood that the hydrocarbon chain is preferably substitutedwith a moiety which forms part of the attachment to the amino acid sidechain or the spacer, for example an acyl group, a sulphonyl group, an Natom, an O atom or an S atom. Most preferably the hydrocarbon chain issubstituted with acyl, and accordingly the hydrocarbon chain may be partof an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoylor stearoyl.

Accordingly, the lipophilic substituent may have the formula shownbelow:

A may be, for example, an acyl group, a sulphonyl group, NH, N-alkyl, anO atom or an S atom, preferably acyl. n is an integer from 3 to 29,preferably at least 7 or at least 11, and preferably 23 or less, morepreferably 19 or less.

The hydrocarbon chain may be further substituted. For example, it may befurther substituted with up to three substituents selected from NH₂, OHand COOH. If the hydrocarbon chain is further substituted, preferably itis further substituted with only one substituent. Alternatively oradditionally, the hydrocarbon chain may include a cycloalkane orheterocycloalkane, for example as shown below:

Preferably the cycloalkane or heterocycloalkane is a six-membered ring.Most preferably, it is piperidine.

Alternatively, the lipophilic substituent may be based on acyclopentanophenanthrene skeleton, which may be partially or fullyunsaturated, or saturated. The carbon atoms in the skeleton each may besubstituted with Me or OH. For example, the lipophilic substituent maybe cholyl, deoxycholyl or lithocholyl.

As mentioned above, the lipophilic substituent may be conjugated to theamino acid side chain by a spacer. When present, the spacer is attachedto the lipophilic substituent and to the amino acid side chain. Thespacer may be attached to the lipophilic substituent and to the aminoacid side chain independently by an ester, a sulphonyl ester, athioester, an amide or a sulphonamide. Accordingly, it may include twomoieties independently selected from acyl, sulphonyl, an N atom, an Oatom or an S atom. The spacer may have the formula:

wherein B and D are each independently selected from acyl, sulphonyl,NH, N-alkyl, an O atom or an S atom, preferably from acyl and NH.Preferably, n is an integer from 1 to 10, preferably from 1 to 5. Thespacer may be further substituted with one or more substituents selectedfrom C₁₋₆ alkyl, C₀₋₆ alkyl amine, C₀₋₆ alkyl hydroxy and C₀₋₆ alkylcarboxy.

Alternatively, the spacer may have two or more repeat units of theformula above. B, D and n are each selected independently for eachrepeat unit. Adjacent repeat units may be covalently attached to eachother via their respective B and D moieties. For example, the B and Dmoieties of the adjacent repeat units may together form an ester, asulphonyl ester, a thioester, an amide or a sulphonamide. The free B andD units at each end of the spacer are attached to the amino acid sidechain and the lipophilic substituent as described above.

Preferably the spacer has five or fewer, four or fewer or three or fewerrepeat units. Most preferably the spacer has two repeat units, or is asingle unit.

The spacer (or one or more of the repeat units of the spacer, if it hasrepeat units) may be, for example, a natural or unnatural amino acid. Itwill be understood that for amino acids having functionalised sidechains, B and/or D may be a moiety within the side chain of the aminoacid. The spacer may be any naturally occurring or unnatural amino acid.For example, the spacer (or one or more of the repeat units of thespacer, if it has repeat units) may be Gly, Pro, Ala, Val, Leu, Ile,Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln, Asn, α-Glu, γ-Glu, Asp, SerThr, Gaba, Aib, β-Ala, 5-aminopentanoyl, 6-aminohexanoyl,7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or 10-aminodecanoyl.

For example, the spacer may be a single amino acid selected from γ-Glu,Gaba, b-Ala and α-Gly.

The lipophilic substituent may be conjugated to any amino acid sidechain in the compounds of the invention. Preferably, the amino acid sidechain includes an carboxy, hydroxyl, thiol, amide or amine group, forforming an ester, a sulphonyl ester, a thioester, an amide or asulphonamide with the spacer or lipophilic substituent. For example, thelipophilic substituent may be conjugated to Asn, Asp, Glu, Gln, His,Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dpr or Orn. Preferably, thelipophilic substituent is conjugated to Lys. However, any amino acidshown as Lys in the formulae provided herein may be replaced by Dbu, Dpror Orn where a lipophilic substituent is added.

An example lipophilic substituent and spacer is shown in the formulabelow:

Here, a Lys from the compound of the present invention (e.g. from X) iscovalently attached to γ-Glu (the spacer) by via an amide moiety.Palmitoyl is covalently attached to the γ-Glu spacer via an amidemoiety.

In a particular embodiment of the present invention, the compoundcomprises or consists of peptide X having the sequence:

(SEQ ID NO: 17) HSQGTFTSDYSKYLDSK(isoGlu(Palm))AAHDFVEWLLRA

Alternatively or additionally, one or more amino acid side chains in thecompound of the invention may be conjugated to a polymeric moiety, forexample, in order to increase solubility and/or half-life in vivo (e.g.in plasma) and/or bioavailability. Such modification is also known toreduce clearance (e.g. renal clearance) of therapeutic proteins andpeptides.

The polymeric moiety is preferably water soluble (amphiphilic orhydrophilic), non-toxic, and pharmaceutically inert. Suitable polymericmoieties include polyethylene glycol (PEG), homo- or co-polymers of PEG,a monomethyl-substituted polymer of PEG (mPEG), or polyoxyethyleneglycerol (POG). See, for example, Int. J. Hematology 68:1 (1998);Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug CarrierSys. 9:249 (1992).

Other suitable polymeric moieties include poly-amino acids such aspoly-lysine, poly-aspartic acid and poly-glutamic acid (see for exampleGombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, etal. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al. (1984),J. Natl. Cancer Inst., vol 73: 721-729; and Pratesi, et al. (1985), Br.J. Cancer, vol. 52: 841-848).

The polymeric moiety may be straight-chain or branched. It may have amolecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000Da, 10,000-20,000 Da, or 20,000-40,000 Da.

A compound may comprise two or more such moieties, in which case thetotal molecular weight of all such moieties will generally fall withinthe ranges provided above.

The polymeric moiety may be coupled (by covalent linkage) to an amino,carboxyl or thiol group of an amino acid side chain. Preferred examplesare the thiol group of Cys residues and the epsilon amino group of Lysresidues, and the carboxyl groups of Asp and Glu residues may also beused.

The skilled reader will be well aware of suitable techniques which canbe used to perform the coupling reaction. For example, a PEG moietycarrying a methoxy group can be coupled to a Cys thiol group by amaleimido linkage using regents commercially available from NektarTherapeutics AL. See also WO 2008/101017, and the references cited abovefor details of suitable chemistry.

Peptide Synthesis

The compounds of this invention may be manufactured either by standardsynthetic methods, recombinant expression systems; or any other state ofthe art method. Thus the glucagon analogues may be synthesized in anumber of ways including for example, a method which comprises:

(a) synthesizing the peptide by means of solid phase or liquid phasemethodology either stepwise or by fragment assembling and isolation andpurification of the final peptide product;

(b) expressing a nucleic acid construct that encodes the peptide in ahost cell and recovering the expression product from the host cellculture; or

(c) effecting cell-free in vitro expression of a nucleic acid constructthat encodes the peptide and recovering the expression product;

or any combination of methods of (a), (b), and (c) to obtain fragmentsof the peptide, subsequently ligating the fragments to obtain thepeptide, and recovering the peptide.

It is preferred to synthesize the analogues of the invention by means ofsolid phase or liquid phase peptide synthesis. In this context,reference is given to WO 98/11125 and, amongst many others, Fields, G Bet al., 2002, “Principles and practice of solid-phase peptidesynthesis”. In: Synthetic Peptides (2nd Edition) and the Examplesherein.

For recombinant expression, the nucleic acid fragments of the inventionwill normally be inserted in suitable vectors to form cloning orexpression vectors carrying the nucleic acid fragments of the invention;such novel vectors are also part of the invention. The vectors can,depending on purpose and type of application, be in the form ofplasmids, phages, cosmids, mini-chromosomes, or virus, but also nakedDNA which is only expressed transiently in certain cells is an importantvector. Preferred cloning and expression vectors (plasmid vectors) ofthe invention are capable of autonomous replication, thereby enablinghigh copy-numbers for the purposes of high-level expression orhigh-level replication for subsequent cloning.

In general outline, an expression vector comprises the followingfeatures in the direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma), the nucleic acid fragment encoding the peptide of theinvention, and optionally a nucleic acid sequence encoding a terminator.They may comprise additional features such as selectable markers andorigins of replication. When operating with expression vectors inproducer strains or cell lines it may be preferred that the vector iscapable of integrating into the host cell genome. The skilled person isvery familiar with suitable vectors and is able to design one accordingto their specific requirements.

The vectors of the invention are used to transform host cells to producethe compound of the invention. Such transformed cells, which are alsopart of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are micro-organisms such asbacteria (such as the species Escherichia (e.g. E. coli), Bacillus (e.g.Bacillus subtilis), Salmonella, or Mycobacterium (preferablynon-pathogenic, e.g. M. bovis BCG), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells may bederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, a plant cell, or a mammalian cell. For the purposes ofcloning and/or optimised expression it is preferred that the transformedcell is capable of replicating the nucleic acid fragment of theinvention. Cells expressing the nucleic fragment are useful embodimentsof the invention; they can be used for small-scale or large-scalepreparation of the peptides of the invention.

When producing the peptide of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is secreted into the culture medium.

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptorsmay be used as an indication of agonist activity, but in general it ispreferred to use a biological assay which measures intracellularsignalling caused by binding of the compound to the relevant receptor.For example, activation of the glucagon receptor by a glucagon agonistwill stimulate cellular cyclic AMP (cAMP) formation. Similarly,activation of the GLP-1 receptor by a GLP-1 agonist will stimulatecellular cAMP formation. Thus, production of cAMP in suitable cellsexpressing one of these two receptors can be used to monitor therelevant receptor activity. Use of a suitable pair of cell types, eachexpressing one receptor but not the other, can hence be used todetermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. The GLP-1 receptor and/or the glucagon receptor mayhave the sequence of the receptors as described in the examples. Forexample, the assays may make use the human glucagon receptor(Glucagon-R) having primary accession number GI: 4503947 and/or thehuman glucagon-like peptide 1 receptor (GLP-1R) having primary accessionnumber GI:166795283. (Where sequences of precursor proteins are referredto, it should of course be understood that assays may make use of themature protein, lacking the signal sequence).

EC₅₀ values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀-[GLP-1R]lower than the EC₅₀-[GLP-1R] of native glucagon in a particular assaymay be considered to have higher potency at the GLP-1R than glucagon.

The compounds described in this specification are typically Glu-GLP-1dual agonists, i.e. they are capable of stimulating cAMP formation atboth the glucagon receptor and the GLP-1 receptor. The stimulation ofeach receptor can be measured in independent assays and afterwardscompared to each other.

By comparing the EC₅₀ value for the glucagon receptor (EC₅₀[Glucagon-R]) with the EC₅₀ value for the GLP-1 receptor, (EC₅₀[GLP-1R]) for a given compound the relative glucagon selectivity (%) ofthat compound can be found:Relative Glucagon-Rselectivity[Compound]=(1/EC₅₀[Glucagon-R])×100/(1/EC₅₀[Glucagon-R]+1/EC₅₀[GLP-1R])

The relative GLP-1R selectivity can likewise be found:Relative GLP-1Rselectivity[Compound]=(1/EC₅₀[GLP-1R])×100/(1/EC₅₀[Glucagon-R]+1/EC₅₀[GLP-1R])

A compound's relative selectivity allows its effect on the GLP-1 orglucagon receptor to be compared directly to its effect on the otherreceptor. For example, the higher a compound's relative GLP-1selectivity is, the more effective that compound is on the GLP-1receptor as compared to the glucagon receptor.

Using the assays described below, we have found the relative GLP-1selectivity for human glucagon to be approximately 5%.

The compounds of the invention have a higher relative GLP-1R selectivitythan human glucagon. Thus, for a particular level of glucagon-R agonistactivity, the compound will display a higher level of GLP-1R agonistactivity (i.e. greater potency at the GLP-1 receptor) than glucagon. Itwill be understood that the absolute potency of a particular compound atthe glucagon and GLP-1 receptors may be higher, lower or approximatelyequal to that of native human glucagon, as long as the appropriaterelative GLP-1R selectivity is achieved.

Nevertheless, the compounds of this invention may have a lower EC₅₀[GLP-1R] than human glucagon. The compounds may have a lowerEC₅₀-[GLP-1-R] than glucagon while maintaining an EC₅₀ [Glucagon-R] thatis less than 10-fold higher than that of human glucagon, less than5-fold higher than that of human glucagon, or less than 2-fold higherthan that of human glucagon.

The compounds of the invention may have an EC₅₀ [Glucagon-R] that isless than two-fold that of human glucagon. The compounds may have anEC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon andhave an EC₅₀ [GLP-1R] that is less than half that of human glucagon,less than a fifth of that of human glucagon, or less than a tenth ofthat of human glucagon.

The relative GLP-1 selectivity of the compounds may be between 5% and95%. For example, the compounds may have a relative selectivity of5-20%, 10-30%, 20-50%, 30-70%, or 50-80%; or of 30-50%, 40-60,%, 50-70%or 75-95%.

Therapeutic Uses

The compounds of the invention may provide an attractive treatmentoption for obesity and metabolic diseases including type 2 diabetes.

Diabetes mellitus, often referred to simply as diabetes, is a syndromeof disordered metabolism, usually due to a combination of hereditary andenvironmental causes, resulting in abnormally high blood sugar levels(hyperglycemia).

Blood glucose levels are controlled by the hormone insulin made in thebeta cells of the pancreas. Diabetes develops due to destruction ofinsulin producing pancreatic beta-cells (in type 1 diabetes) orresistance to the effects of insulin (in gestational diabetes) followedby beta cell loss (in type 2 diabetes). Both types of diabetes lead tohyperglycemia, which largely causes the acute signs of diabetes:excessive urine production, resulting compensatory thirst and increasedfluid intake, blurred vision, unexplained weight loss, lethargy, andchanges in energy metabolism.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person. They include abdominal obesity (excessive fat tissuearound the abdominal internal organs), atherogenic dyslipidemia (bloodfat disorders including high triglycerides, low HDL cholesterol and/orhigh LDL cholesterol, which foster plaque buildup in artery walls),elevated blood pressure (hypertension), insulin resistance and glucoseintolerance, prothrombotic state (e.g. high fibrinogen or plasminogenactivator inhibitor-1 in the blood), and proinflammatory state (e.g.,elevated C-reactive protein in the blood).

Individuals with the metabolic syndrome are at increased risk of type 2diabetes as well as coronary heart disease and other diseases related toother manifestations of arteriosclerosis (e.g., stroke and peripheralvascular disease). The dominant underlying risk factors for thissyndrome appear to be abdominal obesity and insulin resistance andglucose intolerance, prothrombotic state (e.g. high fibrinogen orplasminogen activator inhibitor-1 in the blood), and proinflammatorystate (e.g., elevated C-reactive protein in the blood).

Without wishing to be bound by any particular theory, it is believedthat the compounds of the invention act as GluGLP-1 dual agonists. Thedual agonist combines the effect of glucagon on fat metabolism with theeffects of GLP-1 on blood glucose levels and food intake. They mighttherefore act in a synergistic fashion to accelerate elimination ofexcessive fat deposition, induce sustainable weight loss, and directlydecrease morbid glucose levels to normal levels, without the risk ofhypoglycemia, which is associated with concomitant use of GLP-1 agonistsand sulphonylurea.

The synergetic effect of dual GluGLP-1 agonists may also result inreduction of cardiovascular risk factors such as high cholesterol andLDL, as well as an improvement in glucose tolerance, which may beentirely independent of their effect on body weight.

The compounds of the present invention can therefore be used aspharmaceutical agents for preventing weight gain, promoting weight loss,reducing excess body weight or treating obesity (e.g. by control ofappetite, feeding, food intake, calorie intake, and/or energyexpenditure), including morbid obesity, as well as associated diseasesand health conditions including but not limited to obesity linkedinflammation, obesity linked gallbladder disease and obesity inducedsleep apnea. The compounds of the invention may also be used fortreatment of metabolic syndrome, insulin resistance, glucoseintolerance, type 2 diabetes, hypertension, atherogenic dyslipidimia,atherosclerois, arteriosclerosis, coronary heart disease and stroke.These are all conditions which can be associated with obesity. However,the effects of the compounds of the invention on these conditions may bemediated in whole or in part via an effect on body weight, or may beindependent thereof.

Pharmaceutical Compositions

The compounds of the present invention, or salts thereof, may beformulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the invention, or a salt thereof, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompasses any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to the salt of thecompounds. Salts include pharmaceutically acceptable salts such as acidaddition salts and basic salts. Examples of acid addition salts includehydrochloride salts, citrate salts and acetate salts. Examples of basicsalts include salts where the cation is selected from alkali metals,such as sodium and potassium, alkaline earth metals, such as calcium,and ammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designatesoptionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are described in“Remington's Pharmaceutical Sciences”, 17th edition. Ed. Alfonso R.Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 andmore recent editions, and in the Encyclopaedia of PharmaceuticalTechnology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms: It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Pharmaceutically acceptablecarriers or diluents include those used in formulations suitable fororal, rectal, nasal, topical (including buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous,intradermal, and transdermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the compounds described herein.

Combination Therapy

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity orhypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention (or the salt thereof) can be used incombination with an anti-diabetic agent including but not limited tometformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone,or insulin. In a preferred embodiment the compound or salt thereof isused in combination with insulin, DPP-IV inhibitor, sulfonylurea ormetformin, particularly sulfonylurea or metformin, for achievingadequate glycemic control. In an even more preferred embodiment thecompound or salt thereof is used in combination with insulin or aninsulin analogue for achieving adequate glycemic control. Examples ofinsulin analogues include but are not limited to Lantus, Novorapid,Humalog, Novomix, and Actraphane HM

The compound or salt thereof can further be used in combination with ananti-obesity agent including but not limited to a glucagon-like peptidereceptor 1 agonist, peptide YY or analogue thereof, cannabinoid receptor1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, ormelanin concentrating hormone receptor 1 antagonist.

The analogue compound or salt thereof can be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretics, beta-blocker, or calcium channel blocker.

Methods

General Synthesis of Glucagon Analogues

Solid phase peptide synthesis was performed as SPPS on a microwaveassisted synthesizer using standard Fmoc strategy in NMP on apolystyrene resin (TentaGel S Ram). HATU was used as coupling reagenttogether with DIPEA as base. Piperidine (20% in NMP) was used fordeprotection. Pseudoprolines: Fmoc-Phe-Thr(.Psi. Me, Me pro)-OH andFmoc-Asp-Ser(.Psi., Me, Me pro)-OH (purchased from NovaBiochem) wereused where applicable.

Cleavage:

The crude peptide was cleaved from the resin by treatment with95/2.5/2.5% (v/v) TFA/TIS/water at r.t. for 2 h. For peptides with amethionine in the sequence a mixture of 95/5% (v/v) TFA/EDT was used.Most of the TFA was removed at reduced pressure and the crude peptidewas precipitated and washed with diethylether and allowed to dry toconstant weight at ambient temperature.

HPLC Purification of the Crude Peptide

The crude peptides were purified by standard reverse phase HPLC on aPerSeptive Biosystems VISION Workstation. VISION 3.0 software was usedfor instrument control and data acquisition. The peptides were analysedusing MS and purified to greater than 90% as determined by HPLC.

General Synthesis of Acylated Glucagon Analogues

The peptide backbone is synthesized as described above for the generalsynthesis of glucagon analogues, with the exception that it is acylatedon the side chain of a lysine residue with the peptide still attached tothe resin and fully protected on the side chain groups, except theepsilon-amine on the lysine to be acylated. The lysine to be acylated isincorporated with the use of Fmoc-Lys(ivDde)-OH. The N-terminal of thepeptide is protected with a Boc group using Boc₂O in NMP. While thepeptide is still attached to the resin, the ivDde protecting group isselectively cleaved using 2% hydrazine hydrate in NMP. The unprotectedlysine side chain is then first coupled with a spacer amino acid likeFmoc-Glu-OtBu, which is deprotected with piperidine and acylated with afatty acid using standard peptide coupling methodology as describedabove. Alternatively, the histidine at the N-terminal could beincorporated from the beginning as Boc-His(Boc)-OH. Cleavage from theresin and purification are performed as described above

Analysis of Peptide Stability

The glucagon analogues were incubated as solid compounds at 40° C. anddissolved as solutions in 0.1M aqueous HCl (2 mg/ml). The solutions wereincubated at 40° The remaining intact glucagon analogs were measured atRP-HPLC by integration of the UV signal at 220 nM The percentageremaining is a measure for the relative stability.

The solid and solutions of glucagon compounds were prior to analysisdiluted in HPLC solvent to a concentration of 0.2 mg/mL and analyzed atappropriate time points.

TABLE 1 Analytical HPLC set-up. Column Gemini C18 150 × 3 mm Gradient(0-3 min; 18% B) (3-22 min; 45% B) (time; % B) (22-23 min; 95% B) (23-24min; 18% B) (24-30 min; 18% B) Solvent A 0.1% TFA in 1% MeCN:MQW SolventB 0.085% TFA in MeCN Flow 0.300 mL/min Injection Volume 35 μL ColumnTemp. 30° C. UV detection 220 nmGeneration of Cell Lines Expressing Human Glucagon- and GLP-1Receptors

The cDNA encoding either the human glucagon receptor (Glucagon-R)(primary accession number P47871) or the human glucagon-like peptide 1receptor (GLP-1R) (primary accession number P43220) were cloned from thecDNA clones BC104854 (MGC:132514/IMAGE:8143857) or BC112126(MGC:138331/IMAGE:8327594), respectively. The DNA encoding theGlucagon-R or the GLP-1-R was amplified by PCR using primers encodingterminal restriction sites for subcloning. The 5′-end primersadditionally encoded a near Kozak consensus sequence to ensure efficienttranslation. The fidelity of the DNA encoding the Glucagon-R and theGLP-1-R was confirmed by DNA sequencing. The PCR products encoding theGlucagon-R or the GLP-1-R were subcloned into a mammalian expressionvector containing a neomycin (G418) resistance marker.

The mammalian expression vectors encoding the Glucagon-R or the GLP-1-Rwere transfected into HEK293 cells by a standard calcium phosphatetransfection method. 48 hr after transfection cells were seeded forlimited dilution cloning and selected with 1 mg/ml G418 in the culturemedium. Three weeks later 12 surviving colonies of Glucagon-R andGLP-1-R expressing cells were picked, propagated and tested in theGlucagon-R and GLP-1-R efficacy assays as described below. OneGlucagon-R expressing clone and one GLP-1-R expressing clone were chosenfor compound profiling.

Glucagon Receptor and GLP-1-Receptor Efficacy Assays

HEK293 cells expressing the human Glucagon-R, or human GLP-1-R wereseeded at 40,000 cells per well in 96-well microtiter plates coated with0.01% poly-L-lysine and grown for 1 day in culture in 100 μl growthmedium. On the day of analysis, growth medium was removed and the cellswashed once with 200 μl Tyrode buffer. Cells were incubated in 100 μlTyrode buffer containing increasing concentrations of test peptides, 100μM IBMX, and 6 mM glucose for 15 min at 37° C. The reaction was stoppedby addition of 25 μl 0.5 M HCl and incubated on ice for 60 min. The cAMPcontent was estimated using the FlashPlate® cAMP kit from Perkin-Elmer.EC₅₀ and relative efficacies compared to reference compounds (glucagonand GLP-1) were estimated by computer aided curve fitting.

Lipolysis in Primary Rat Adipocytes

The effect of glucagon analogues on lipolysis was assessed in primarycultures of rat adipocytes. Adipocytes were isolated from epididymal fatdissected from normal young adult Sprague-Dawley rats. The fat lumpswere minced, incubated and shaken (220 rpm) with collagenase (1 mg/ml)in Krebs-Ringer buffer containing 4% BSA (KRB-BSA) for 60 minutes at 37°C. The suspension was filtered through a nylon filter (160 μm pore size)and the filtrate centrifuged at 200×g for 3 min. The subjacent mediumbeneath the upper floating layer of adipocytes was removed with aPasteur-pipette. The adipocytes were washed 3 times in KRB-BSA buffer byre-suspension and centrifugation. The adipocytes were re-suspended inKRB-BSA, mixed, incubated in and shaken with test compounds in 96-deepwell plates (50,000 cells/well) in a total volume of 1 ml at 37° C. for60 min. The plates were placed on ice for at least 10 min afterincubation followed by centrifugation at 200×g for 3 min. 300 μl of thebuffer beneath the adipocyte layer were collected in a 96-deep wellplate. This process was repeated two more times and the 3 extractscollected from each culture pooled together. The glycerol formed by thelipolysis in the adipocyte cultures was measured by adding free glycerolreagent (200 μl) to aliquots (25 μl) of adipocyte extract, incubate atroom temperature for 15 min and measure the absorbance at 540 nm.

Oral Glucose Tolerance Test (OGTT) in db/db Mice

Db/db mice were fasted overnight and an initial blood sample (fastingblood glucose level) taken just before administration (i.p.) of vehicle(Acetate buffer, 20 mM acetic acid, 250 mM mannitol, pH 5.0) or the testcompound ZP2669 (SEQ ID NO: 4) (45 nmol/kg in acetate buffer). Theanimals were kept fasted during the experiment to prevent confoundingfood intake. Fifteen minutes later an oral dose of glucose (1 g/kg in 5ml/kg) was given and BG levels were measured at t=30 min, t=60 min,t=120 min and t=240 min.

Difference from baseline (t=0) was calculated for each time point andAUC_(0-240 min) values were determined. Statistical analyses of AUCvalues by one-way ANOVA and Dunnetts post-hoc analyses were performedwith GraphPad Prism version 4. Differences were considered significantat the p<0.05 level.

Food Intake in Normal Mice

One week before study C57BI/6 mice (8 weeks) (N=9-12 animals in eachgroup) were conditioned to treatment by daily injections (s.c.) of 0.2ml vehicle and acclimatized to handling by weighing them twice a week.Mice were stratified one day before start of experiment into groups withsimilar body weight (BW). Groups of stratified mice were fastedovernight treated (10 μl test solution/g BW, s.c.) with PYY₃₋₃₆ (30nmol/kg)(internal positive control), glucagon (500 nmol/kg) and ZP2669(SEQ ID NO: 4) (500 nmol/kg) or vehicle. After one hour pre-weighed foodwas introduced to the mice and food intake measured by weighing theremaining food after one hour and expressed relative to body weight (mgfood/g BW). Statistical analyses of food intake data by one-way ANOVAand Dunnetts post-hoc analyses were performed with GraphPad Prismversion 4. Differences were considered significant at the p<0.05 level.

Effects of 28 Day Treatment of Diet Induced Obese (D10) Mice with ZP2669(SEQ ID NO: 4) Agonist on Body Weight Gain and Plasma Cholesterols

Four weeks before drug treatments, C57BI/6 male mice (7 weeks)) (N=10-12animals in each group) were put on high fat diet (HFD) and theirday-night cycle reversed with lights On/Off at 2000/0800 hour.Experimental animals were conditioned to treatment by daily injections(s.c.) of 0.1 ml vehicle and acclimatized to handling by weighing themtwice a week one week before start of drug administrations. The daybefore start of experiment, mice were stratified into groups withsimilar body weight (BW) and the next day groups of stratified mice weretreated (b.i.d.; s.c.) with ZP2669 (SEQ ID NO: 4) (500 nmol/kg) orvehicle (Acetate-buffer). ZP2669 (SEQ ID NO: 4) was given in isotonicacetate buffer (pH 4.8; 2.5 μl/g BW). A non-obese control groupmaintained on regular chow was treated with vehicle in the sametreatment regime as the DIO groups. Body weights were recorded daily andused to administer the body weight-corrected doses of peptide throughoutthe study. Animals were fasted overnight before sacrifice. An eye bloodsample (0.6 ml EDTA) was obtained the following morning immediatelybefore cervical dislocation. Blood plasma samples were stored at −80° C.until analyzed for cholesterol, HDL and LDL using commercially availablekits. Body weight gains throughout the treatment period were calculatedfor each animal by subtracting its weight at the initiation oftreatment.

The effects of treatment on body weight gain and cholesterols wereassessed by 2-way ANOVA with repeated measures and Bonferoni post-hocanalyses, using GraphPad Prism version 4. Differences were consideredsignificant at the p<0.05 level.

Results Example 1 Peptide Stability

TABLE 2 Results for recovery measured by RP-HPLC after stress. ZPRecovery (%) Recovery (%) Compound Solid peptide 0.1M HCl Glucagon 91 152669 98 58

The results of incubation in HCl stress solutions are shown in table 2.Both the analogue and glucagon were stable over 5 weeks at 40° C. with arecovery of over 90% purity. However, the results of the acidicdegradation of the compounds shows that the glucagon analogue is almostfour times more stabile than native glucagon.

Example 2 Efficacy on Glucagon and GLP-1Receptors

TABLE 3EC₅₀ values of glucagons analogues at Glucagon and GLP-1 receptorsGLP-1R GLUR. EC₅₀ EC₅₀ (nM) (nM) Glucagon 20 0.10 OXM 1.0 0.50 Exendin-40.02 >1,000. ZP2669 H-HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-NH2 0.05 0.05(SEQ ID NO: 4) ZP2668 H-HSQGTFTSDYSKYLDSKAARDFVRWLKLA-NH2 0.08 0.80(SEQ ID NO: 5) ZP2866 H-H-DSer-QGTFTSDYSKYLDSKAAHDFVEWLLRA- 0.09 0.11(SEQ ID NH2 NO: 6) ZP2867 H-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA-NH2 0.080.06 (SEQ ID NO: 7) L006-0171 H-HSQGTFTSDYSKYLDSKAAQDFVEWLLRA-NH2 0.370.78 (SEQ ID NO: 8)* L006-0172 H-HSQGTFTSDYSKYLDSKAAHDFVQWLLRA-NH2 0210.37 (SEQ ID NO: 9)* L006-0176 H-HSQGTFTSDYSKYLDSKAAHDFVEWLMNT-NH2 0.440.57 (SEQ ID NO: 10)* L006-0212 H-HSQGTFTSDYSKYLDSKAAHDFVKWLLRA-NH2 0.761.23 (SEQ ID NO: 11)* L006-0169 H-HSQGTFTSDYSKYLDSRAAHDFVEWLLRA-NH2 0.99152 (SEQ ID NO: 12)* L006-0170 H-HSQGTFTSDYSKYLDSKRAHDFVEWLLRA-NH2 0.500.79 (SEQ ID NO: 13)* ZP2753 H-HSQGTFTSDYSKYLDSLAAQDFVQWLLNT-NH2 0.040.08 (SEQ ID NO: 14) ZP2667 H-HSQGTFTSDYSKYLDSKAAQDFVQWLMNT-NH2 0.020.07 (SEQ ID NO: 15) ZP2872 H- 0.103 0.085 (SEQ IDHSQGTFTSDYSKYLDSK(isoGlu(Palm))AAHDFVEWLL NO: 17) RA-NH2 L006-0208H-HSQGTFTSDYSKYLDSKAAKDFVEWLLRA-NH2 0.57 0.55 (SEQ ID NO: 18)* L006-0239H-HSQGTFTSDYSKYLDKKAAHDFVEWLLRA-NH2 0.98 1.05 (SEQ ID NO: 19)* L006-0296H-HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-OH 0.14 0.12 (SEQ ID NO: 20)* Thecompounds marked “*” are crude peptides with purity of less than 90%.The EC50 is corrected to a purity of 50%. IsoGlu(Palm) conjugated to alysine residue is illustrated in FIG. 6.

Example 3 Lipolysis Assay

TABLE 4 Stimulation of lipolysis in primary rat adipocyte cultures (fordetails see Methods). Compound EC50 (nM) Exendin-4 No effect Glucagon 6OXM 180 ZP2669 4.6 (SEQ ID NO: 4) ZP2668 101 (SEQ ID NO: 5) ZP2667 0.75(SEQ ID NO: 15)

The GLP-1 agonist, exendin-4 had no effect on lipolysis in primaryadipocyte cultures in contrast to glucagon and OXM.ZP2667 was 8 times aspotent as glucagon and 240 times as potent as OXM, while ZP2669 (SEQ IDNO: 4) was equipotent with glucagon and 40 times as potent as OXM. Thepotency of ZP2668 (SEQ ID NO: 5) was in the same order of magnitude asOXM. The finding that 4 weeks treatment of DIO mice with ZP2669 (SEQ IDNO: 4) significantly decreased fat deposition concurs with the effectobserved (Table 4) on lipid metabolism in primary adipocyte cultures.

Example 4 Effect on Oral Glucose Tolerance in db/db Mice

ZP2669 (SEQ ID NO: 4) significantly (P<0.01) improved glucose tolerancemeasured during an OGTT in diabetic db/db mice (FIG. 1). ZP2669 (SEQ IDNO: 4) improved glucose tolerance by 77.1% at 45 nmol/kg.

Example 5 Effect on Food Intake in Mice

The vehicle treated animals ate 0.033±0.001 g food/g body weight duringthe first hour (FIG. 2). PYY(3-36) showed an anorectic effect asexpected from previous findings. ZP2669 (SEQ ID NO: 4) (500 nmol/kg)significantly (P<0.05) decreased food intake during the first hourfollowing injection of peptide (FIG. 2). Glucagon had no effect on foodintake.

Example 6 Effect of Subcutaneous Administration on Body Weight Gain inDiet Induced Obese Mice

ZP2669 (SEQ ID NO: 4) decreased body weight gain to a level similar tothat observed with chow feeding (FIG. 3). The body weight gain wasstatistically significantly (P<0.05) less than the vehicle group.

Example 7 Effects on LDL and HDL

Exendin-4 is a very potent GLP-1R agonist, but has no effect on the GIuRand has no effect in the rat adipocyte lipolysis assay described above.Furthermore, Exendin-4 has no effect on blood concentrations of totalcholesterol, HDL, LDL or HDL/LDL ratios (FIGS. 4, 5)

In contrast, treatment of DIO mice with ZP2669 (SEQ ID NO: 4) had asignificant effect on blood concentration of LDL cholesterol (P=0.0006)as well as HDL/LDL ratios (P=0.0005) compared to vehicle (FIGS. 4, 5).

The invention claimed is:
 1. A compound having the formula R¹-X-Z-R²wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂; X is a peptide which has the formula I: (SEQ ID NO: 4)His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-Arg-Ala

or differs from formula I at up to 4 of the following positions whereby,if different from formula I: the residue at position 2 is selected from:Aib, D-Ser; the residue at position 16 is Lys; the residue at position17 is selected from: Arg, Leu; the residue at position 18 is Arg; theresidue at position 20 is selected from: Gln, Lys, Arg; the residue atposition 24 is selected from: Gln, Lys, Arg; the residue at position 27is selected from: Met, Lys; the residue at position 28 is selected from:Asn, Leu; and the residue at position 29 is Thr; and Z is absent or apeptide sequence of 1-20 amino acid units selected from the groupconsisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr andOrn; or a pharmaceutically acceptable salt thereof.
 2. A compound havingthe formula R¹-X-Z-R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl,benzoyl or trifluoroacetyl; R² is OH or NH₂; X is a peptide which hasthe formula I: (SEQ ID NO: 4)His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-Arg-Ala

or differs from formula I at up to 5 of the following positions whereby,if different from formula I: the residue at position 2 is selected from:Aib, D-Ser; the residue at position 16 is Lys; the residue at position17 is selected from: Arg, Leu; the residue at position 20 is selectedfrom: Gln, Lys, Arg; the residue at position 24 is selected from: Gln,Lys, Arg; the residue at position 27 is selected from: Met, Lys; theresidue at position 28 is selected from: Asn, Leu; and the residue atposition 29 is Thr; and Z is absent or a peptide sequence of 1-20 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or a pharmaceuticallyacceptable salt thereof.
 3. A compound according to claim 1 wherein Xdiffers from formula I at up to 4 of the following positions whereby, ifdifferent from formula I: the residue at position 2 is selected from:Aib, D-Ser; the residue at position 16 is Lys; the residue at position24 is selected from: Gln, Lys, Arg; the residue at position 27 isselected from: Met, Lys; the residue at position 28 is selected from:Asn, Leu; and the residue at position 29 is Thr.
 4. A compound accordingto claim 1 wherein X comprises one or more of the following groups ofresidues: (a) 17-Lys, 18-Ala; (b) 17-Leu, 18-Ala; (c) 17-Lys, 18-Ala,20-His; (d) 17-Leu, 18-Ala, 20-His; (e) 17-Lys, 18-Ala, 24-Glu; (f)17-Leu, 18-Ala, 24-Glu; (g) 17-Lys, 18-Ala, 27-Leu; (h) 17-Leu, 18-Ala,27-Leu; (i) 17-Lys, 18-Ala, 29-Ala; (j) 17-Leu, 18-Ala, 29-Ala; (k)17-Lys, 18-Ala, 27-Leu, 29-Ala; (l) 17-Leu, 18-Ala, 27-Leu, 29-Ala; (m)17-Lys, 18-Ala, 27-Leu, 28-Arg, 29-Ala; (n) 17-Leu, 18-Ala, 27-Leu,28-Arg, 29-Ala; (o) 24-Glu, 28-Arg; (p) 24-Glu, 28-Arg, 27-Leu; (q)24-Glu, 28-Arg, 27-Leu, 29-Ala; (r) 27-Leu, 28-Arg, 29-Ala; (s) 29-Ala;(t) 20-Arg, 24-Arg, 27-Lys, 28-Leu; (u) 17-Arg; (v) 18-Arg; (w) 20-Gln;(x) 24-Gln; (y) 27-Met, 28-Asn, 29-Thr; or (z) 24-Lys.
 5. A compoundaccording to claim 2 wherein X comprises one of the following groups ofresidues: (a) 20-Gln, 24-Gln, 27-Met, 28-Asn, 29-Thr; or (b) 17-Leu,20-Gln, 24-Gln, 28-Asn, 29-Thr.
 6. A compound according to claim 1wherein X has the sequence: (SEQ ID NO: 6)H-DSer-QGTFTSDYSKYLDSKAAHDFVEWLLRA; (SEQ ID NO: 7)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA; (SEQ ID NO: 5)HSQGTFTSDYSKYLDSKAARDFVRWLKLA; (SEQ ID NO: 12)HSQGTFTSDYSKYLDSRAAHDFVEWLLRA; (SEQ ID NO: 13)HSQGTFTSDYSKYLDSKRAHDFVEWLLRA; (SEQ ID NO: 8)HSQGTFTSDYSKYLDSKAAQDFVEWLLRA; (SEQ ID NO: 9)HSQGTFTSDYSKYLDSKAAHDFVQWLLRA; (SEQ ID NO: 10)HSQGTFTSDYSKYLDSKAAHDFVEWLMNT; (SEQ ID NO: 11)HSQGTFTSDYSKYLDSKAAHDFVKWLLRA; (SEQ ID NO: 18)HSQGTFTSDYSKYLDSKAAKDFVEWLLRA; (SEQ ID NO: 19)HSQGTFTSDYSKYLDKKAAHDFVEWLLRA; or (SEQ ID NO: 20)HSQGTFTSDYSKYLDSKAAHDFVEWLLRA.


7. A compound according to claim 2 wherein X has the sequence:(SEQ ID NO: 15) HSQGTFTSDYSKYLDSKAAQDFVQWLMNT or (SEQ ID NO: 14)HSQGTFTSDYSKYLDSLAAQDFVQWLLNT.


8. A compound according to claim 1 wherein R¹ is H.
 9. A compoundaccording to claim 1 wherein R² is NH₂.
 10. A compound according toclaim 1 wherein Z has no more than 25% sequence identity with thecorresponding portion of the IP-1 sequence of human oxyntomodulin havingthe sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala.
 11. A compound accordingto claim 1 wherein Z has a Cys as the C-terminal residue.
 12. A compoundaccording to claim 1 wherein Z is absent.
 13. A compound according toclaim 1 wherein one or more of the amino acid side chains in thecompound is conjugated to a lipophilic substituent or a polymericmoiety.
 14. A nucleic acid encoding a compound according to claim
 1. 15.An expression vector comprising a nucleic acid according to claim 14.16. A host cell comprising a nucleic acid according to claim 15 or anexpression vector according to claim
 15. 17. A pharmaceuticalcomposition comprising a compound according to claim 1, in admixturewith a pharmaceutically acceptable carrier.
 18. A method of reducingweight gain, promoting weight loss, or for treatment of a condition,said condition being morbid obesity, obesity linked inflammation,obesity linked gallbladder disease and obesity induced sleep apnea, orfor treatment of insulin resistance, glucose intolerance, type 2diabetes, said method comprising administering a therapeuticallyeffective amount of a compound according to claim
 1. 19. A compoundaccording to claim 13, wherein one or more of the amino acid side chainsin peptide X is conjugated to said lipophilic substituent or polymericmoiety.
 20. A compound according to claim 19 wherein X has the sequence:(SEQ ID NO: 17) HSQGTFTSDYSKYLDSK*AAHDFVEWLLRA

wherein K* indicates a lysine side chain conjugated to isoGlu(Palm). 21.A pharmaceutical composition comprising a nucleic acid according toclaim 14, in admixture with a pharmaceutically acceptable carrier.
 22. Apharmaceutical composition comprising an expression vector according toclaim 15, in admixture with a pharmaceutically acceptable carrier.
 23. Apharmaceutical composition comprising a host cell according to claim 16,in admixture with a pharmaceutically acceptable carrier.
 24. Thecompound of claim 1, wherein X has the sequence: (SEQ ID NO: 6)H-DSer-QGTFTSDYSKYLDSKAAHDFVEWLLRA.


25. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 7)H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA.


26. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 5)HSQGTFTSDYSKYLDSKAARDFVRWLKLA.


27. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 12)HSQGTFTSDYSKYLDSRAAHDFVEWLLRA.


28. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 13)HSQGTFTSDYSKYLDSKRAHDFVEWLLRA.


29. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 8)HSQGTFTSDYSKYLDSKAAQDFVEWLLRA.


30. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 9)HSQGTFTSDYSKYLDSKAAHDFVQWLLRA.


31. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 10)HSQGTFTSDYSKYLDSKAAHDFVEWLMNT.


32. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 11)HSQGTFTSDYSKYLDSKAAHDFVKWLLRA.


33. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 18)HSQGTFTSDYSKYLDSKAAKDFVEWLLRA.


34. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 19)HSQGTFTSDYSKYLDKKAAHDFVEWLLRA.


35. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 20)HSQGTFTSDYSKYLDSKAAHDFVEWLLRA.


36. The compound of claim 2, wherein X has the sequence: (SEQ ID NO: 15)HSQGTFTSDYSKYLDSKAAQDFVQWLMNT.


37. The compound of claim 1, wherein X has the sequence: (SEQ ID NO: 14)HSQGTFTSDYSKYLDSLAAQDFVQWLLNT.